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Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

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(A) Model description of the steady state gating charge movement. The averaged Q(V) curves for the wf, wfF401L, and wfF401A channels are replotted from Fig. 9. Fits of the model in Fig. 14 are shown superimposed as solid lines. These were obtained by integrating the simulated OFF gating currents at −100 mV, low-pass filtered at 10 kHz, following steps to the test potentials shown on the x axis in 2-mV increments and normalizing by the charge obtained at the most positive voltages. This analysis follows the normal procedure we use for the experimental data. (B) Comparison of gating current kinetics with the model predictions. Representative families of gating currents from oocytes containing wf (top, from −80 to +40 mV), wfF401L (middle, −80 to +40 mV), and wfF401A (bottom, −95 to +25 mV) channels are shown. Voltage increment is 20 mV in each case. Simulated gating currents are superimposed (thin lines). Models shown in Fig. 14 were used for the three channels, with the following modifications to obtain better fits to these experimental families: in the wf model, λ0 = 101 s−1; in the F401L model, λ0 = 39 s−1; and in the F401A model, κ0 = 3,500 s−1, λ0 = 8,000 s−1, and zγ = 0.8 e0.
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Figure 15: (A) Model description of the steady state gating charge movement. The averaged Q(V) curves for the wf, wfF401L, and wfF401A channels are replotted from Fig. 9. Fits of the model in Fig. 14 are shown superimposed as solid lines. These were obtained by integrating the simulated OFF gating currents at −100 mV, low-pass filtered at 10 kHz, following steps to the test potentials shown on the x axis in 2-mV increments and normalizing by the charge obtained at the most positive voltages. This analysis follows the normal procedure we use for the experimental data. (B) Comparison of gating current kinetics with the model predictions. Representative families of gating currents from oocytes containing wf (top, from −80 to +40 mV), wfF401L (middle, −80 to +40 mV), and wfF401A (bottom, −95 to +25 mV) channels are shown. Voltage increment is 20 mV in each case. Simulated gating currents are superimposed (thin lines). Models shown in Fig. 14 were used for the three channels, with the following modifications to obtain better fits to these experimental families: in the wf model, λ0 = 101 s−1; in the F401L model, λ0 = 39 s−1; and in the F401A model, κ0 = 3,500 s−1, λ0 = 8,000 s−1, and zγ = 0.8 e0.

Mentions: Fig. 15 A shows the fits of the model shown in Fig. 14 for the wf, wfF401L, and wfF401A channel's steady state charge vs. voltage curves. Equilibrium constants for the two charge-moving transitions in each subunit and for the concerted step were optimized to obtain the desired steepness and position along the voltage axis. The total charge displacement for a given transition is the sum of the charges that move before and after the transition state or, equivalently, that are associated with the forward and backward rates of that transition. The three channels differ the most in their equilibria for the concerted opening transition. The zero-voltage equilibrium constants for this step are 55, 125, and 0.5 for the wf, wfF401L, and wfF401A, respectively. The marked decrease in wfF401A provides part of the explanation for the shallowness of the slope of its Q(V) curve, even though this transition carries only ∼1/16 of the total charge displacement in the channel. To describe accurately the relatively shallow lower portion of each of the curves, it is necessary to make the charge displacement associated with the second of the two sequential subunit transitions (zγδ) greater than that of the first (zαβ) (Bezanilla et al. 1994). For all channel species, the quantity zγδ is 2.0–2.1 e0. The first transition carries the charge of 1.35 e0. Models for wfF401L and wfF401A channels make the zero-voltage equilibrium constant for the first transition, \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}K_{0{\mathrm{{\alpha}{\beta}}}}=\frac{k_{0{\mathrm{{\alpha}}}}}{k_{0{\mathrm{{\beta}}}}}{\mathrm{,}}\end{equation*}\end{document} approximately twice as great, and that for the second transition, \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}K_{0{\mathrm{{\gamma}{\delta}}}}=\frac{k_{0{\mathrm{{\gamma}}}}}{k_{0{\mathrm{{\delta}}}}}{\mathrm{,}}\end{equation*}\end{document} nearly three times as great as those of the wf model in order to account for the more negative voltage range over which the initial gating charge movement occurs in the mutants.


Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

(A) Model description of the steady state gating charge movement. The averaged Q(V) curves for the wf, wfF401L, and wfF401A channels are replotted from Fig. 9. Fits of the model in Fig. 14 are shown superimposed as solid lines. These were obtained by integrating the simulated OFF gating currents at −100 mV, low-pass filtered at 10 kHz, following steps to the test potentials shown on the x axis in 2-mV increments and normalizing by the charge obtained at the most positive voltages. This analysis follows the normal procedure we use for the experimental data. (B) Comparison of gating current kinetics with the model predictions. Representative families of gating currents from oocytes containing wf (top, from −80 to +40 mV), wfF401L (middle, −80 to +40 mV), and wfF401A (bottom, −95 to +25 mV) channels are shown. Voltage increment is 20 mV in each case. Simulated gating currents are superimposed (thin lines). Models shown in Fig. 14 were used for the three channels, with the following modifications to obtain better fits to these experimental families: in the wf model, λ0 = 101 s−1; in the F401L model, λ0 = 39 s−1; and in the F401A model, κ0 = 3,500 s−1, λ0 = 8,000 s−1, and zγ = 0.8 e0.
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Figure 15: (A) Model description of the steady state gating charge movement. The averaged Q(V) curves for the wf, wfF401L, and wfF401A channels are replotted from Fig. 9. Fits of the model in Fig. 14 are shown superimposed as solid lines. These were obtained by integrating the simulated OFF gating currents at −100 mV, low-pass filtered at 10 kHz, following steps to the test potentials shown on the x axis in 2-mV increments and normalizing by the charge obtained at the most positive voltages. This analysis follows the normal procedure we use for the experimental data. (B) Comparison of gating current kinetics with the model predictions. Representative families of gating currents from oocytes containing wf (top, from −80 to +40 mV), wfF401L (middle, −80 to +40 mV), and wfF401A (bottom, −95 to +25 mV) channels are shown. Voltage increment is 20 mV in each case. Simulated gating currents are superimposed (thin lines). Models shown in Fig. 14 were used for the three channels, with the following modifications to obtain better fits to these experimental families: in the wf model, λ0 = 101 s−1; in the F401L model, λ0 = 39 s−1; and in the F401A model, κ0 = 3,500 s−1, λ0 = 8,000 s−1, and zγ = 0.8 e0.
Mentions: Fig. 15 A shows the fits of the model shown in Fig. 14 for the wf, wfF401L, and wfF401A channel's steady state charge vs. voltage curves. Equilibrium constants for the two charge-moving transitions in each subunit and for the concerted step were optimized to obtain the desired steepness and position along the voltage axis. The total charge displacement for a given transition is the sum of the charges that move before and after the transition state or, equivalently, that are associated with the forward and backward rates of that transition. The three channels differ the most in their equilibria for the concerted opening transition. The zero-voltage equilibrium constants for this step are 55, 125, and 0.5 for the wf, wfF401L, and wfF401A, respectively. The marked decrease in wfF401A provides part of the explanation for the shallowness of the slope of its Q(V) curve, even though this transition carries only ∼1/16 of the total charge displacement in the channel. To describe accurately the relatively shallow lower portion of each of the curves, it is necessary to make the charge displacement associated with the second of the two sequential subunit transitions (zγδ) greater than that of the first (zαβ) (Bezanilla et al. 1994). For all channel species, the quantity zγδ is 2.0–2.1 e0. The first transition carries the charge of 1.35 e0. Models for wfF401L and wfF401A channels make the zero-voltage equilibrium constant for the first transition, \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}K_{0{\mathrm{{\alpha}{\beta}}}}=\frac{k_{0{\mathrm{{\alpha}}}}}{k_{0{\mathrm{{\beta}}}}}{\mathrm{,}}\end{equation*}\end{document} approximately twice as great, and that for the second transition, \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}K_{0{\mathrm{{\gamma}{\delta}}}}=\frac{k_{0{\mathrm{{\gamma}}}}}{k_{0{\mathrm{{\delta}}}}}{\mathrm{,}}\end{equation*}\end{document} nearly three times as great as those of the wf model in order to account for the more negative voltage range over which the initial gating charge movement occurs in the mutants.

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

Show MeSH
Related in: MedlinePlus